The body of a normal adult contains 4.0 to 5.5 liters of blood, composed of approximately 60% fluid (plasma) and 40% formed elements (red cells; white cells; and platelets). Under normal physiological conditions, the main function of the platelets is prevention of hemorrhage (bleeding).
Platelets are formed in the bone marrow from precursor cells called megakaryocytes and have a life span of 8 to 10 days in circulation. As a consequence of this short life span, platelet deficiency occurs rapidly when the ability of the bone marrow to produce platelets is depressed, such as in cancer patients undergoing chemotherapy. Platelet deficiency also may occur as a result of various diseases, for example when antibodies are produced in vivo against platelet surface glycoproteins or against other platelet surface antigens. Such depletion or destruction of platelets results in an insufficient quantity of circulating platelets (a condition referred to as thrombocytopenia) and can cause uncontrolled bleeding. Platelet deficiency also may result from surgery involving for example extracorporeal circulation which tends to damage or destroy circulating platelets. Clinical management of thrombocytopenia typically has involved transfusion of fresh, intact platelets.
It is predominantly held that only metabolically-active, intact platelets can function in vivo to arrest bleeding. As a consequence, transfusion therapy has been dependent on the procurement of high quality, fresh, viable platelets. Platelets for transfusion typically are prepared: (a) from freshly donated blood units, called random-donor platelets or (b) from a single donor by apheresis, called single-donor platelets. These transfusion products are plasma suspensions of fresh, concentrated, intact platelets.
A major drawback of the current practice of platelet transfusion is the short shelf life of intact platelets, three to five days. Many platelet units collected by hospitals, bloodbanks and the like unfortunately are discarded due to outdating. Because of their short shelf life, it is very difficult to maintain large inventories of platelet units. This problem is particularly critical in connection with decentralized operations, such as the military, civil defense and disaster agencies.
Several substitutes for intact, viable platelets have been attempted for transfusion both clinically and in animal models. In 1956 (1), it was reported that hemostasis was achieved following the clinical administration of lyophilized platelets, suggesting that the morphological integrity of platelets may not be essential for the retention of at least some of the in vivo functions of intact platelets. A major side effect of the intravenous administration of the lyophilized platelet material was that the patient experienced severe pain at the site of infusion, possibly due to vasospasm caused by the high serotonin content in the lyophilized material. Contrary to the foregoing result, it was reported in 1959 (2) that fresh, ultrasound-disrupted whole platelet preparations failed to reduce the number of erythrocytes in the lymph of thrombocytopenic dogs. More recently, McGill et al (3) reported that the transfusion of platelet membrane concentrates shortened bleeding times in thrombocytopenic rabbits. The concentrate included ghost platelets about the size of a normal platelet and containing mitochondria and remnants of the surface-connecting system. McGill's concentrate was prepared by: (1) centrifuging the whole blood of rabbits to pellet fresh platelets; (2) freezing the pellet at -65.degree. C.; (3) thawing and then twice--freezing and thawing the pellet; and (4) rinsing and resuspending the pellet in platelet-free plasma.
In preparing the foregoing platelet membrane fractions, temperature conditions of about 4.degree. C. or below were maintained. Such temperatures are standard when working with biologicals as activity is routinely lost in very short intervals when biologicals are exposed to higher temperatures. For example, proteins such as enzymes may be inactivated by heating them to about 60.degree. C. Activity may be lost even at 4.degree. C. For example, it has been reported that partially purified platelet factor 3 (PF-3) loses a major portion of its clotting activity after five days storage at 4.degree. C. (4). (PF-3 appears to be associated with a platelet membrane complex that provides a catalytic surface to promote thrombin generation.) Such loss of activity is of great concern when considering the use of a platelet fraction as a pharmaceutical.
When preparing a platelet fraction for use in humans, it is of course necessary to use sterile conditions. Like whole blood transfusions, the use of donated platelet units exposes the recipients to the risk of transmission of diseases such as AIDS, hepatitis, and other transfusion-related diseases. Another risk is that after multiple transfusions, the recipient/patients may develop antibodies against donated platelets (a condition known as alloimmunization). Such antibodies can cause rapid destruction of the platelets transfused. Further, bacterial contamination of stored platelets is a significant hazard in transfusions.